KLF4-PFKFB3-driven glycolysis is essential for phenotypic switching of vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) within atherosclerotic lesions undergo a phenotypic switching in a KLF4-dependent manner. Glycolysis plays important roles in transdifferentiation of somatic cells, however, it is unclear whether and how KLF4 mediates the link between glycolytic switch and VSMCs phenotypic transitions. Here, we show that KLF4 upregulation accompanies VSMCs phenotypic switching in atherosclerotic lesions. KLF4 enhances the metabolic switch to glycolysis through increasing PFKFB3 expression. Inhibiting glycolysis suppresses KLF4-induced VSMCs phenotypic switching, demonstrating that glycolytic shift is required for VSMCs phenotypic switching. Mechanistically, KLF4 upregulates expression of circCTDP1 and eEF1A2, both of which cooperatively promote PFKFB3 expression. TMAO induces glycolytic shift and VSMCs phenotypic switching by upregulating KLF4. Our study indicates that KLF4 mediates the link between glycolytic switch and VSMCs phenotypic transitions, suggesting that a previously unrecognized KLF4-eEF1A2/circCTDP1-PFKFB3 axis plays crucial roles in VSMCs phenotypic switching.

16. Data on Apoe ko mice must be better presented. Usually, to have consistency among different animals, the tissue sections must be obtained close to the aortic valve, where usually plaque form. Furthermore, to draw conclusions, a statistical quantification must be also enclosed together with the representative images. 17. Data presented in figure 8d must be completed with the labeling of CD123. 18. We strongly suggest thoroughly proofread the manuscript to improve the overall quality of the scientific communication. Interesting manuscript with well-performed experiments and solid data. However, a few remarks need to be addressed: -While the manuscript is in general well-written, the coherence between sentences, in particular the introduction, is lacking. Especially between the different paragraphs in the introduction addressing all the different previous findings (circRNAs,TMAO,metabolism,KLF4,CD123). This makes the introduction hard to read. While in the result resection the structure appears to be there. Please rewrite the introduction, since placing the information into context would increase the readability of the manuscript.
-Why did the authors focus on CD123? Please introduce this as well.
-Please provide the quantification of the pixel overlap between the markers used for confocal imaging analysis (eg. Fig 1C and S1).
-Could the authors explain the partial expression of CD123 in the nucleus in Fig 1G? -Does reduction in KLF4 expression (siRNA) reduce CD123 expression? -Please include glucose levels in medium as well, besides measuring lactate levels to solidify the glycolysis data.
-The authors should include their main findings in the histological plaques as well (e.g confocal microscopy and stain for PFKFB3 expression in combination with KLF4)) -To further test whether VSMC-derived pDC-like cells were able to produce interferon alpha (IFN-α), which is an important function of maturated pDCs, we treated KLF4-overexpressing VSMCs with CpG oligodeoxynucleotides (ODN), an inducer of production of IFN-α, and detected IFN-α production This sentence needs to be rewritten, since it's stated at the end that IFNa production was detected, it insinuates there is a difference between ctrl and KLF4-OE cells.
-Does a knock-down of PFKFB3 in KLF4 over-expressing cells affect CD123 expression? This question came up when reading the first PFKFB3 knock-down experiments, however this question is answered in Figure 6. Please move this data up.
Reviewer #3 (Remarks to the Author): <B>What are the major claims of the paper? Are they novel and will they be of interest to others in the community and the wider field? If the conclusions are not original, it would be helpful if you could provide relevant references. <B> Atherosclerosis is a major underlying cause of cardiovascular related death and understanding how to ameliorate or prevent adverse complications from atherosclerosis, including plaque rupture or erosion, is a major focus of preclinical and clinical research. Historically, much of this focus has been on reducing cholesterol, leading to the widespread use of statins, and on detrimental inflammation. Recently, smooth muscle cells have become a target to increase plaque stabilization, and in the process, metabolic state and regulation has emerged as a major player in smooth muscle cell phenotypic switching and ECM deposition. The authors expand on previous work detailing the importance of metabolic state in atherosclerosis, and in SMC in particular. In this manuscript, the authors provide evidence that SMC to pDC phenotypic switching is regulated by KLF4, PFKFB3, eEF1A2, and circCTFP1. While the authors clearly layout the role KLF4 plays in regulating SMC metabolic state in vitro, this was first shown, although not in as much detail, in previous work by Alencar et al (Circ 2020) where knockout of KLF4 in SMC showed significant changes in metabolic pathways by bulk RNAseq, as well as resulting in SMC modulation based on immunofluorescence and scRNAseq analyses. Other relevant papers in the field of atherosclerosis, metabolism, and SMC include: Newman et al Nature Metab 2021 (SMC phenotypic transitions and ECM synthesis are regulated by metabolic state), Tomas et al EHJ 2018 (metabolism in symptomatic vs non-symptomatic lesion), and the multiple papers from the 1980s including Lynch and Paul Experientia 1985 (energy metabolism and contractility ability in SMC). <B>The novelty of this manuscript stems mostly from the identification of the small non coding RNA effectors of phenotypic switching, especially as it relates to post-RNA or post-translational modification of protein as a way to induce phenotypic changes via modulating metabolic state. This is quite exciting and this idea in particular will be important for the eventual goal of targeting individual cell types in disease. However, the conclusions about phenotypic state as well as impact on atherosclerosis in this paper are not entirely supported by the data. Nevertheless, identification of this RNA modifier axis has the potential to influence thinking with substantial clarifications and/or revisions.</B> <B>Is the work convincing, and if not, what further evidence would be required to strengthen the conclusions?<B> The authors outline putative SMC to pDC phenotypic switching and correlate that to atherosclerotic lesion progression. The SMC to pDC switch itself is, to this reviewer's knowledge, unknown previously. However, the identification of SMC-derived pDCs is solely reliant on marker protein co-localization of SMC marker proteins and pDC marker proteins. This is problematic for a number of reasons including the ubiquity of many of the markers used by the authors in atherosclerotic lesions, the propensity of SMC themselves to downregulate their own marker proteins (rendering use of ACTA2 in vivo unreliable), and the relative inabundance of pDCs in atherosclerotic lesions (as cited in ref 32). Since this is a main point the authors are trying to make, it is imperative that evidence for this be incontrovertible.
Major comments: 1. SMC to pDC phenotypic switching. To this reviewer, the almost complete co-localization of ACTA2 staining with CD123 (pDC marker) is suspect as authors seem to imply that all ACTA2+ (putative SMC) have transitioned to a pDC phenotype. Couple this with the multiple SMC scRNAseq studies over the past few years (Wirka et al, Pan et al, Alencar et al) that do not show a pDC-like cluster derived from murine sorted lineage tagged SMC, this reviewer is not convinced of the author's claims of SMC to pDC transition in atherosclerosis. Further, there is no attempt to positively identify the ACTA2+ cells as SMC. This reviewer suggests confirming SMC origin by more than ACTA2 expression as well as providing more substantial evidence than marker staining that pDC-like cells exist, as this is the crux of the author's story. While the reviewer is aware that lineage tracing is impossible in humans, claims of SMC-derived pDC can be supported by either In Situ Hybridization with Proximity Ligation Assay (Gomez et al Nat Meth 2012) and/or use of the many murine SMC-lineage tracing models available. 2. pDC specificity marker CD123. The idea of SMC can differentiate to a pDC-like cell is bolstered by the in vitro work showing increased CD123 and other pan DC mRNA expression (sometimes 3000fold!) after KLF4 OE, however, authors do not evaluate any other marker of pDC (except for pan CD11c in mice) in vivo nor functionality aside from increased IFNa expression. Considering CD123 in disease is also used as a marker for hematopoietic stem cells, the reviewer suggests including additional metrics to determine a pDC-like state in vivo. Authors may consider including data using KLF4 KD vs. wild type in order to understand if this occurs normally, or is a function of the OE. 3. IF staining controls. The reviewer questions the settings used for analysis of co-localization in Figures 1 and 8 and Supp Figs 1 and 5. The images presented look over-processed and/or over-blown. This especially apparent in the media and the body of the lesion where there is an unexpected morphology of the ACTA2 staining as well as the seemingly 100% co-localization of multiple markers including CD68, CD123, and ACTA2. Most previous reports, including ones the author cites (reference 4) show that about 10% of the lesion cells are ACTA2+ and that only a very small fraction of ACTA2+ cells co-stain for macrophage markers. The reviewer urges the authors to show the unprocessed images and to provide images of negative controls and more detailed information about assessment of co-localization and relative abundance of cell types in the methods. 4. Quantification of claims: a. Please quantify the percentage of ACTA2 cells that co-localize with the various markers in the figures (e.g. what is one to understand from "…obvious reduction in the expression of these genes"? page 9 line 345). Please also quantify plaque metrics to bolster claims of "enhance[d] atherosclerosis". b. The reviewer suggests including a description of and relative abundance of pDC in plaques. The authors suggest that an increase in ACTA2+ CD123+ cells in atherosclerotic lesions in mice is associated with increased plaque size (without quantification), but aside from this, there is no clear stated directionality on if these metabolic changes are beneficial or detrimental, nor if changes (purportedly increases) in pDC-like cells are protective or pro-atherogenic (excluding plaque size, which is not the best indicator of stability, for references see the works of Virmani et al from CVPath). 5. Conclusions. Because of the lack of lineage tracing, quantification of lesion area, cell phenotype, and negative controls for immunofluorescence images, the authors are suggested to tone down a number of the major conclusions in the paper. a. On Page 10 paragraph 1, the authors suggest that ACTA2 staining is indicative of a SMC origin, but do not take into account the fact that in mice, nearly 80% of the SMC-derived cells have lost their ACTA2+ marker expression, especially those in the lesion core (ref 4). b. In SFig 1, authors note that "pDC marker CD123 and macrophage marker CD68 are abundantly expressed in VSMCs…" indicating that CD123 and CD68 are co-expressed, but the idea that the pDC are derived from monocytes/macrophages is not explored. Indeed, monocytes and pDC share a known common progenitor (HSC, MPP), whereas SMC and pDC do not share a known progenitor, is it possible that 1. The pDC in the lesion are derived from the CD68 cells (i.e. myeloid cells that may have acquired ACTA2 expression, Newman Nat Metab 2021, Albarran-Juarez Atherosclerosis 2016, Caplice PNAS 2003) or 2. the co-localization of these three markers in the plaque is an artifact of staining? c. The role of KLF4 in SMC phenotypic switching is well-known, however the authors suggest that KLF4 exclusively regulates the SMC to pDC switch. Indeed, Alencar et al (Circ 2020) show a number of KLF4-regulated SMC-derived phenotypes in atherosclerosis including a chondrocyte-like cell and other osteogenic phenotypes but not a pDC-like phenotype. Nor is this shown in the other related scRNAseq studies of SMC-labeled cells in atherosclerosis (Wirka et al, Pan et al). Conclusions would be supported if authors show this unique KLF4-PFKFB3-RNA axis is specific for pDC transitions.
Minor comments: 1. Please define PFKFB3 page 4 line 107 2. Figure 1c, SFig 1a, SFig 5: please indicate if the field of view is in the core or the cap region of the lesion 3. Please add graphs quantifying the overlap of ACTA2 cells that co-localize with the various markers in the figures (i.e. what is one to understand from "…obvious reduction in the expression of these genes"? page 9 line 345) 4. Please add detail to the methods about cell culture conditions including duration of treatments 5. Please ensure conclusions are borne out in the data or clarify/tone down conclusions not directly show in the manuscript. For example: clarify claim on pg 11 line 429 that the "…glycolytic shift…can also activate Akt" by showing these metabolic using Seahorse with glycolysis agonists and KLF4 OE (refer to page 8 line 286, where it "evidently" causes changes). 6. Regarding Akt expression in atherosclerosis pathogenesis, authors show Akt is phosphorylated after KLF4 OE as part of the PFKFB3 axis and later suggest that this accelerates atherosclerosis pathology. A seminal paper in the field (Fernandez-Hernando ATVB 2009) shows Akt1 is required for SMC survival and for overall plaque stabilization. How do the authors claims fit in with the wider field? 7. The authors should include in the figure legends statistical tests performed, error bars (SEM or SD?), replicate numbers, and scale bars for all images to facilitate assessment of statistical analysis and validity of data.

Point-by-point response to reviewer 1
In the present manuscript, Zhang and colleagues investigated the role of the axis KLF4-PFKFB3 in the phenotypic switch of VSMCs. Via an integrated approach, the authors tried to highlight a very complex pathway in which a transcription factor, a translation modulator, a circular RNA, and a metabolic enzyme are involved. This is a effort, however, due to the complexity of the analysis, the paper falls short in different aspects, that must be further investigated. Below a specific review for the authors:

Q1:
The authors stated that KLF4 triggers a switch of VSMCs toward a pCD-like phenotype. In my opinion, the authors must provide further data showing that this is happening. First, in the analyzed plaques (figure 1), there is no co-staining KLF4-CD68-ACTA2, this would really suggest that such transition might happen. Then, in general, all immunofluorescence data in the paper must be accompanied by quantification and statistical analysis using different samples, with sufficient power analysis.

A1:
Thank you for this helpful suggestion. We performed the co-staining experiments for KLF4/CD123/ACTA2 and KLF4/CD68/ACTA2 using a multiplex tyramide signal amplification (TSA) staining, which is open and flexible for use with any primary antibody without cross-reactivity. The results showed that pDC marker CD123 and macrophage marker CD68 were colocalized with VSMCs in the analyzed plaques (Please refer to Figs. 1c, d and Supplementary Fig. 1). Experimental methods and results were described, respectively, in Method and Result section. As a result, we observed 19% and 12% of SM α-actin positive (SMA-α + ) cells that expressed CD123 and CD68, respectively, in human atherosclerotic lesions. Importantly, all the CD123 + SMA-α + and CD68 + SMA-α + cells were KLF4 positive. All immunofluorescence data have been quantified and statistically analyzed accordingly.
Q2: About the KLF4-GFP construct used for the gain-of-function experiments, is it a bicistronic vector of it generates a fusion KLF4-GFP? This is important because when it expresses KLF4 the GFP localization is mainly nuclear.

A2:
The KLF4-GFP construct used for the gain-of-function experiments generates a fusion KLF4-GFP. GFP was fused to the C-terminal of KLF4 cDNA, the termination codon of KLF4 was removed, and then the fusion fragment was cloned into adeno-associated virus plasmid for adeno-associated virus packaging, with the promoter as CMV.

Q3:
The conclusion reached with the experiments reported in the first paragraph is not totally supported, because, although the modulation of gene expression might suggest a switch due to KLF4, no metabolic/biological variations are observed (IFN production).
Therefore, all structure of the work is limited by that and not fully supported, thus these functional data are essential.

A3:
We thank the reviewer's constructive comment. Because producing abundant interferon alpha (IFN-α) upon stimulation by foreign nucleic acids is a unique capacity of pDCs, we tested whether KLF4-overexpressing cells treated with CpG oligodeoxynucleotides (ODN) were able to produce IFN-α in the previous manuscript.
Results showed that IFN-α-producing activities were hardly detectable in the KLF4-overexpressing VSMCs, and thus they remain distinguishable from the authentic pDCs. Consistently, a subset of studies showed that SMC-derived cells within atherosclerotic lesions express multiple markers of other cells, such as macrophages (Mϕs) and mesenchymal stem cells (MSCs), but they did not appear to function as statements. For example, the two sentences "These findings suggest that VSMCs within the plaques switch to a pDC-like phenotype and that KLF4 upregulation is correlated with VSMC phenotypic transitions" and "Taken together, these results indicate that KLF4 upregulation is responsible for the switching of VSMCs to pDC-like phenotype" in the previous manuscript were summarized into one sentence "These findings suggest that VSMCs within the plaques may switch to a pDC-like phenotype and that KLF4 upregulation is correlated with this phenotypic change" (page 4 line 133).

Q4:
The functional data about the modulation of glycolysis in KLF4-overpressing cells are interesting, but does the contrary happen in loss-of-function conditions? If so, will the authors be able to perform a rescue experiment with cells transduced with siRNAs vs KLF4 and a construct expressing a not targetable KLF4 of a different origin, such as mouse one?

A4:
We have performed the loss-of-function experiments with siRNA vs KLF4, however, knockdown of KLF4 showed little effect on lactate production and PFKFB3 expression (data not shown and Fig. 7o), although silencing KLF4 suppressed TMAO-induced expression of PFKFB3 (Fig. 7o). A possible reason is that KLF4 overexpression might reach supra-physiological levels. However, the dose of adenovirus had been titrated and tracked relative to endogenous KLF4 levels before using. On the other hand, the specificity of gene expression modulated by KLF4 overexpression was confirmed by the fact that KLF5 overexpression did not affect these gene expressions (Figs. 1f and 4g).

Q5:
Is there, in KLF4-expressing VSMCs, an alteration of the expression of fundamental glycolytic genes, such as HKII?
A5: Western blot analysis was performed to detect the expression of HK2 in KLF4-overexpressing VSMCs. Consistent with the mRNA microarray analyses of KLF4-overexpressing VSMCs and the TMT-based LC-MS/MS data (Fig. 3a, b), overexpression of KLF4 in VSMCs did not affect HK2 expression (Fig. 3d, e).

Q6:
In the WB for eEF1A2 (figure 4) there are two bands, but only one is reduced in presence of specific siRNAs, what is the other one?
A6: As the reviewer raised, we also observed there are two bands in the WB for eEF1A2 when performed experiments. We analyzed that the band with lower molecular weight is nonspecific with nearly the same quantity in each group. It does not seem to be the other subunit of eEF1A, eEF1A1, because the molecular weight of the two isoforms is almost the same. Importantly, the efficacy and specificity of siRNAs had been also confirmed by qRT-PCR, showing a significant reduction of eEF1A2 mRNA level (75%) but not the eEF1A1 mRNA (data not shown).

Q7:
The link between the different studied molecular pathways and circRNAs is a very long shot, what prompted the authors to study that? A7: We and others observed that circRNAs play fundamental roles in cardiovascular diseases through their miRNA/protein-binding capacity (Circ Res, 2017,121:628-635;Nucleic Acids Res, 2019,47:3580-3593;Theranostics, 2017,7:3842-3855). Thus, we focused the coactivator for eEF1A2 on circRNAs and performed circRNA microarrays to screen differentially expressed circRNAs regulated by KLF4 and established a method to construct circRNA overexpression vector (Patent No. ZL201710215532.0). Q8: Did the authors experimentally validate the identified circRNAs? I see only qPCR data, and while circCTDP1 was already reported and validate by others, none is available on circZFAT. There are pipelines of wet experiments that will help to demonstrate whether potential circRNAs are really circular.

A8:
The presence of circZFAT and circCTDP1 had been validated by using divergent primers to amplify circRNAs formed by head-to-tail splicing. We added the data to the Supplementary figure section in the revised version ( Supplementary Fig. 7). A10: The physical co-localization of circCTDP1 and eEF1A2 in cells has been confirmed by a combined in situ/protein staining in the revised version (Fig. 5j).

Q11:
The authors must also measure the protein level of PFKFB3 in cells treated with 2-DG and PFK15.

A11:
When the activities of glycolytic enzymes, hexokinase and PFKFB3, were inhibited by 2-DG and PFK15, respectively, PFKFB3 expression was not altered as demonstrated by Western blot analysis in the revised version ( Supplementary Fig. 10a). Thus, we drawed the conclusion that "This implies that the effect of STAT3 activation on CD123 expression is cell-context dependent".

Q13:
For the TMAO experiments, the conclusion at page 8 line 307 is not supported by experimental evidence. The authors must show that TMAO alters the biology of cells with specific functional assays.

A13:
In the revised version, we supplemented the experiment to observe the effect of TMAO on IFN-α production in VSMCs. As expected, TMAO treatment could not induce IFN-α production ( Supplementary Fig. 11c), although it could induce CD123 expression and inhibited SM22α and SM α-actin expression (Figs. 7c, g and Supplementary Fig. 11a,   b). Just as we mentioned in response to Q3, we toned down this conclusive statement and revised it as "These results indicated that TMAO could convert VSMCs to a dysfunctional pDC-like cell" (page 8 line 294 and page 8 line 308).
Q14: Then, when using siRNAs vs KLF4, the results must be validated with at least two different oligos, in order to be sure that the observed phenotype is not due to off-targets.
For these experiments, it is clear that the authors used only one siRNA (the best among the two tested), thus the same comment refers also to all reported experiments. As an alternative to using two different siRNAs, the author can also perform a rescue experiment in which the silenced gene is re-expressed using a species analog (i.e. human vs mouse).
Q16: Data on Apoe ko mice must be better presented. Usually, to have consistency among different animals, the tissue sections must be obtained close to the aortic valve, where usually plaque form. Furthermore, to draw conclusions, a statistical quantification must be also enclosed together with the representative images.

A16:
As the reviewer suggested, mouse aortic root paraffin sections were stained using immunofluorescence staining and the immunofluorescence data have been quantified and statistically analyzed (Figs. 8 and Supplementary Fig. 12d-f).
Q17: Data presented in figure 8d must be completed with the labeling of CD123.
A17: CD123 is a marker known to be expressed on human pDCs but not mouse DCs, whereas CD11c is mainly expressed on mouse DCs and pDCs, thus, we stained CD11c using the mouse vessel.

Q18:
We strongly suggest thoroughly proofread the manuscript to improve the overall quality of the scientific communication. The statistical indication for siSTAT3 in Fig. 6g in the previous manuscript has been added in the revised version (Fig. 6f).

Point-by-point response to reviewer 2 Reviewer #2 (Remarks to the Author):
Interesting manuscript with well-performed experiments and solid data. However, a few remarks need to be addressed: Q-While the manuscript is in general well-written, the coherence between sentences, in particular the introduction, is lacking. Especially between the different paragraphs in the introduction addressing all the different previous findings (circRNAs, TMAO, metabolism, KLF4, CD123). This makes the introduction hard to read. While in the result resection the structure appears to be there. Please rewrite the introduction, since placing the information into context would increase the readability of the manuscript.
A: As the reviewer suggested, we rewrote the introduction and made it more coherent and concise. It should be noted that we introduced PFKFB3 in the Introduction section in the revised version according to reviewer 3's comments.

Q-Why did the authors focus on CD123? Please introduce this as well.
A：Because in addition to macrophages (Mϕs) and mesenchymal stem cells (MSCs) markers, VSMCs in human atherosclerotic lesions also express CD123, a pDC marker, and CD123-positive pDCs have been previously detected in atherosclerotic plaques by electron microscopy and immunohistochemistry (Grassia et al, Pharmacol Ther, 2013, 137:172-182;Daissormont et al, Circ Res, 2011, 109:1387-1395. Thus, it is likely that VSMCs also have the potential to switch to DCs. However, little is known about this switch and the role that KLF4 plays in this switch during atherogenesis. Thus, we focused on CD123 and KLF4 regulation of CD123 expression. As the reviewer suggested, we introduced this in the Introduction section in the revised version (page 3 line 64).
Q-Please provide the quantification of the pixel overlap between the markers used for confocal imaging analysis (eg. Fig 1C and S1). Also, according to the reviewer 1's comment to present data on Apoe ko mice with the tissue sections obtained close to the aortic valve (data was from the carotid artery of mouse in the previous manuscript), we performed immunofluorescence staining using mouse aortic root paraffin sections and the immunofluorescence data have been quantified and statistically analyzed accordingly (Figs. 8 and Supplementary Fig. 12d-f). Fig 1G? A: We considered that it might be a result of the high expression of CD123 induced by KLF4 overexpression and supra-physiological levels of CD123 result in its partial nuclear distribution.

Q-Does reduction in KLF4 expression (siRNA) reduce CD123 expression?
A: We had performed the loss-of-function experiments with siRNA vs KLF4, however, knockdown of KLF4 showed little effect on CD123 expression. A possible reason is that KLF4 overexpression might reach supra-physiological levels. However, the dose of adenovirus had been titrated and tracked relative to endogenous KLF4 levels before using.
On the other hand, the specificity of gene expression modulated by KLF4 overexpression was confirmed by the fact that KLF5 overexpression did not affect these gene expressions ( Figs. 1f and 4g). We reasoned that CD123 expression in control VSMCs is extremely low and almost undetectable (Figure 1). Thus, its expression was easily induced but could not be reduced experimentally.
Q-Please include glucose levels in medium as well, besides measuring lactate levels to solidify the glycolysis data.
A: Thank you for this good suggestion. We measured the glucose levels in medium (Fig.   2b) in the revised version. Experimental method and result were described in Method and Result section, respectively.

Q-
The authors should include their main findings in the histological plaques as well (e.g confocal microscopy and stain for PFKFB3 expression in combination with KLF4)) A: We thank the reviewer's constructive comment. The co-staining for KLF4/PFKFB3/SMA-α was performed in human atherosclerotic plaques using a multiplex tyramide signal amplification (TSA) staining (Figure 3l). As a result, PFKFB3 was mainly localized in the nucleus, consistent with the previous report (Li et al, Nat Commun, 2018;9:508) and largely co-localized with KLF4 in the atherosclerotic lesion.
Q-To further test whether VSMC-derived pDC-like cells were able to produce interferon alpha (IFN-α), which is an important function of maturated pDCs, we treated KLF4-overexpressing VSMCs with CpG oligodeoxynucleotides (ODN), an inducer of production of IFN-α, and detected IFN-α production This sentence needs to be rewritten, since it's stated at the end that IFNa production was detected, it insinuates there is a difference between ctrl and KLF4-OE cells.

Point-by-point response to reviewer 3
Reviewer #3 (Remarks to the Author): What are the major claims of the paper? Are they novel and will they be of interest to others in the community and the wider field? If the conclusions are not original, it would be helpful if you could provide relevant references.
Atherosclerosis is a major underlying cause of cardiovascular related death and understanding how to ameliorate or prevent adverse complications from atherosclerosis, including plaque rupture or erosion, is a major focus of preclinical and clinical research.
Historically, much of this focus has been on reducing cholesterol, leading to the widespread use of statins, and on detrimental inflammation. Recently, smooth muscle cells have become a target to increase plaque stabilization, and in the process, metabolic state and regulation has emerged as a major player in smooth muscle cell phenotypic switching and ECM deposition. The authors expand on previous work detailing the importance of metabolic state in atherosclerosis, and in SMC in particular. In this manuscript, the authors provide evidence that SMC to pDC phenotypic switching is regulated by KLF4, PFKFB3, eEF1A2, and circCTFP1. However, the conclusions about phenotypic state as well as impact on atherosclerosis in this paper are not entirely supported by the data. Nevertheless, 0identification of this RNA modifier axis has the potential to influence thinking with substantial clarifications and/or revisions.
Is the work convincing, and if not, what further evidence would be required to strengthen the conclusions?
The authors outline putative SMC to pDC phenotypic switching and correlate that to atherosclerotic lesion progression. The SMC to pDC switch itself is, to this reviewer's knowledge, unknown previously. However, the identification of SMC-derived pDCs is solely reliant on marker protein co-localization of SMC marker proteins and pDC marker proteins. This is problematic for a number of reasons including the ubiquity of many of the markers used by the authors in atherosclerotic lesions, the propensity of SMC themselves to downregulate their own marker proteins (rendering use of ACTA2 in vivo unreliable), and the relative inabundance of pDCs in atherosclerotic lesions (as cited in ref 32). Since this is a main point the authors are trying to make, it is imperative that evidence for this be incontrovertible.   Mol Immunol, 2020,126:25-30). In addition, Ly6D was largely induced by KLF4 overexpression in our study (Fig. 1e, f). Thus, we evaluated Ly6D expression in human atherosclerosis lesion and observed a co-staining of the MYH11 promoter H3K4dime PLA and Ly6D ( Supplementary Fig. 2c, d).

Major comments
We had performed the loss-of-function experiments with siRNA vs KLF4, however, knockdown of KLF4 showed little effect on CD123 expression. A possible reason is that KLF4 overexpression might reach supra-physiological levels. However, the dose of adenovirus had been titrated and tracked relative to endogenous KLF4 levels before using.
We reasoned that CD123 expression in control VSMCs was extremely low (Fig. 1) and could hardly be detected. Thus, its expression was easily induced but could not be reduced experimentally. In addition, the specificity of KLF4-induced pDC marker expressions was confirmed by the fact that KLF5 overexpression did not affect these markers (Fig. 1f).   Fig. 2a, b). Accordingly, the conclusion was revised as "Consistently, we here also observed CD123 expression in human atherosclerotic plaques and a co-localization of CD123 with VSMCs (page 10 line 367)" b. In SFig 1, authors note that "pDC marker CD123 and macrophage marker CD68 are abundantly expressed in VSMCs…" indicating that CD123 and CD68 are co-expressed, but the idea that the pDC are derived from monocytes/macrophages is not explored.
Indeed, monocytes and pDC share a known common progenitor (HSC, MPP), whereas SMC and pDC do not share a known progenitor, is it possible that 1.  (6):628-37). It is not excluded that the VSMCs-derived plaque cells may share the common markers, including the well-known Mϕs marker, chondrocyte marker and the less-known DC marker, which needs to be addressed in future studies. Whether the KLF4-PFKFB3-RNA axis is specific for pDC transitions will be investigated in future work.

A5:
We have rewritten this sentence as "Seahorse glycolysis stress tests were performed and we observed that KLF4 overexpression could increase the extracellular acidification rate (ECAR) related to both glycolysis and glycolytic capacity (Fig. 2e, f)

Q7:
The authors should include in the figure legends statistical tests performed, error bars (SEM or SD?), replicate numbers, and scale bars for all images to facilitate assessment of statistical analysis and validity of data.